Mucosal epithelia form the external interface of many sensitive tissues. The outer surface of cells in these epithelia displays a glycoprotein calyx and adsorbs mucins to create a mucus layer that protects those tissues from abrasion and maintains their hydration. In the cornea, for example, the mucus layer retains the tear film that keeps our eyes wet. Defects in the mucosal layer of the human cornea cause dry eye diseases (DED). These conditions are common, painful, and progress with age. DED is often accompanied by dry mouth, suggesting that its underlying causes affect properties common to mucosal epithelia. One such property is the presence of elaborate actin-based structures on the surface of these epithelial cells, known as microplicae and microridges. These structures have been little studied, but likely make a vital contribution to the functional properties of mucosal epithelia by increasin the surface area of the glycocalyx, thus maximizing their ability to hydrate tissues. Most studies of DED pathology have focused on tear production, but given the critical role of mucosal epithelia in maintaining the mucus layer and tear film, it is likely that defects in epithelial morphogenesis also contribute to these conditions. One of the main obstacles to studying the morphogenesis of microplicae and microridges has been the lack of an accessible model system. We have developed the larval zebrafish skin as a model for studying mucosal epithelial development. The entire surface of zebrafish larvae is wrapped in a single-layered mucosal epithelium known as the periderm. The apical surface of periderm cells is covered my microplicae and microridges that remarkably resemble structures on the surface of the human cornea. These cells are exceptionally accessible to transgenic labeling and confocal imaging, making it possible to visualize the formation of ridges in living animals. Moreover, the amenability of the zebrafish system to sophisticated genetic and transgenic manipulations will make it possible to uncover the underlying molecular mechanisms of microridge formation. In this proposal we combine descriptive, hypothesis-driven, and discovery-based approaches to dissect the process of ridge morphogenesis. Specifically, in Aim 1 we will use live imaging to describe the initial formation of microridges and their re-organization during cellular contraction In Aim 2 we will use molecular and imaging approaches to test the hypothesis that phosphoinositide microdomains orchestrate the formation of microridges. Finally, in Aim 3 we will use RNA-Seq to identify BAR domain proteins and actin regulators enriched in periderm and test whether select candidate proteins play roles in microridge development. Together these studies will provide the first insights into ridge morphogenesis and establish the zebrafish model as a system for understanding not only ridge formation in normal cells, but also the causes of their pathology in diseases, such as DED.

Public Health Relevance

The mucosal epithelia covering many parts of our bodies (including the cornea, conjunctiva, esophagus, pharynx and gut) maintain tissue hydration and protect those tissues from abrasion and infection. Painful dry eye diseases (DED) are caused by defects in hydration of the cornea but few studies have explored the role that epithelial morphogenesis plays in DED. This proposal develops a zebrafish model for studying the morphogenesis of mucosal epithelia in a living animal, providing a promising setting for exploring potential causes of DED and other diseases that afflict mucosal epithelia.